In the human erythrocyte, phosphatidylinositol is phosphorylated to phosphatidylinositol phosphate (PI-P) and phosphatidylinositol-4,5-Biphosphate (PI-P2) by two ATP dependent kinase reactions; these phosphorylated derivatives are converted back to phosphatidylinositol by phosphates which are only partially described. This phosphorylation/dephosphorylation cycle has been implicated in the maintenance of red cell shape, membrane protein mobility, and as a significant consumer of red cell ATP. The goals of this proposal involve understanding the enzymology of phosphatidylinositol phosphorylation/dephosphorylation in the red cells of normals and of patients with hemolytic anemia. Specific goals include purification and characterization of phosphatidylinositol kinase; characterization of phosphatidylinositol phosphate kinase; and examination of the phosphatase activities responsible for the degradation of PI-P and PI-P2. The physiological function of these phosphoinositides in the erythrocyte will also be a major focal point of this proposal. Hydrolysis of PI-P and PI-P2 in the erythrocyte results in the formation of echinocytes from these cells rather than the normal biconcave discs. These findings will be extended to determine the importance of phosphoinositides in the maintenance of cellular shape by erythrocytes. This will include the study of effectors which modify the levels of PI-P and PI-P2 as well as examination of the interaction of phosphoinositides with the erythrocyte cytoskeleton. In addition, it has been reported that up to 70% of the net ATP utilized by the erythrocyte may be consumed in the phosphatidylinositol phosphorylation/dephosphorylation cycle; these findings will be examined to determine the implications of such a massive ATP utilization for this cycle. Erythrocytes from patients with hemolytic anemia are being screened for abnormalities involved in the phosphatidylinositol metabolism pathways; these patients will be very useful in delineating the physiological function of phosphoinositides.